Previous work with the mitochondrial F1F0 ATP synthase from Saccharomyces cerevisiae identified two proteins (Atp11p and Atp12p), whose apparent function is to chaperone the formation of the F1 moiety. Yeast that are defective for either of these "assembly factors" accumulate the F1 alpha and beta subunits in aggregates instead of forming the enzyme oligomer. Although evidence suggests that Atp11p and Atp12p act at a similar point in the F1 assembly pathway, the individual activities of these "assembly factors" are different as indicated by the fact that only Atp11p appears to interact directly with the F1 alpha and beta subunits. One objective of our work is to determine if the actions of Atp11p and Atp12p are principally to protect the alpha and beta subunits from aggregating, or if the assembly factors have a more activate role and actually promote the formation of alphabeta hetero-oligomers. Also, it is of interest to determine if Atp11p and Atp12p exert their activities with the folder or with the unfolded form of the F1 subunits. These issues will be investigated using in vitro assays with purified Atp11p, Atp12p, and F1 alpha and beta subunits. Light scattering measurements will be used to follow F1 protein aggregation in the absence/presence of added effectors, and fluorescence resonance energy transfer (FRET) experiments will be done to monitor hetero-oligomer formation between alpha and beta subunits (starting from the folded or from the unfolded state) that are labeled with differed fluorophores. In other work, the use of cleavable chemical cross-linking reagents coupled with mass spectral peptide mapping, and also genetic assays (yeast two-hybrid, phage display), will be used to map the regions of interactions on Atp11p and on the alpha and beta subunits. To probe questions related to the temporal sequence of Atp11p and Atp12p actives, pulse-chase experiments will be done to follow F1 alphaBeta subunit assembly in vivo in yeast that are conditional for Atp11p and Atp12p activities. Information relevant to the sequence of protein-protein interactions during F1 assembly will come also from work in which complexes formed with F1 alpha and beta subunits that are labeled with photactivatable groups will be followed in time, when the proteins are imported into mitochondria in vitro.